We intend to isolate and purify plasma membrane tumor-associated antigen(s) (TAA) of human osteosarcoma from biopsied and autopsied tumor tissues and from osteosarcoma cell lines. Plasma membrane TAA will be isolated using various solubilization techniques (papain, 6 M guanidine MC1, 0.5% NP40, and sonication). Antigenically active, isolated plasma membrane TAA will be further purified by a combination of methods including DEAE anion-exchange chromatography, Sephadex G-200 gel filtration, preparative polyacrylamide gel electrophoresis, and affinity chromatography. Purified plasma membrane TAA will be analyzed by analytical gel electrophoresis, and isoelectric focusing. Antigenic activities of the isolated and purified plasma membrane TAA will be determined by complement fixation assay, lymphocyte stimulation assay, blocking assay, leukocyte migration inhibition assay, and skin testing of osteosarcoma patients, immunodiffusion, and coprecipitation assays. Monoclonal antibodies to OSAA were prepared by two methods: (1)\hybridomas; and (2)\EBV-infected B lymphocytes from osteosarcoma patients. From hybridomas we obtained two clones which produced specific antibodies to OSAA. For the EBV-infected B lymphocytes procedure, five clones were detected to produce OSAA-specific antibodies. A radioimmunoassay (RIA) will be developed using the monoclonal antibodies for the determination of antigen levels in the sera of osteosarcoma patients. The clinical applications of the RIA will be examined by comparing antigen levels in the sera of osteosarcoma patients at different stages of disease (including those with and without metastases and those in remission), patients with nonneoplastic bone diseases, and normal individuals.